DEAMINATION OF ASPARTIC ACID BY PROTEUS X-19
III. EFFECT OF K + , Na+ AND pH ON DEAMINATION BY WHOLE CELLS
by PHILIP A. TRUDINGER
(From the Department of Biochemistry, University oi Adelaide).
(Accepted for publication 31st August, 1953.)
During an investigation into the effects of inhibitory materials on aspartic
acid deamination by Proteus X-19, it was noted that dipicrylamine produces a
marked depression of ammonia output. Since dipicrylamine is notable for its
ability to combine with potassium ions, a study of the effect of the latter on the
deamination reaction was carried out. It was found that not only potassium
ions, but also sodium ions are necessary for the deamination of aspartic acid
by whole Proteus cells. These requirements had not been detected previously,
since the medium used in the investigation of aspartie acid deamination contained both metals in optimal amounts (Trudinger, 1951).
MATERIALS AND METHODS.
The organism and general protedure have been described in an earlier paper (Trudinger,
1951).
The abbreviated description of buiFers used in this paper ig as followa:
K:Na
K:K
Na:Na
Na borate
K borate
:
:
:
:
:
KH^PO. + NajHPO,
KHjPO. + K.HPO,
NalljPO, + Na^HPO.
Boric acid + NaOH -|~ NaCl
Boric acid + KOH + KCl
In experiments on the influence of pH on the reaction rate, KCl or NaCl was added
to the respective bufFpr solutions so that the K* or Na* concentrations remained the same over
the complete pH range.
Neutralization of substrate, etc., was carried out with the metal hydroxide corresponding
to the buffer. pH determinations were carried out ou a Jones Model B electrometer. Sodium
and potassium were determined on the Beckmann Model DU spectrophotometer with flame
attachment (Wynn et al., 1950). Pumarate was estimated by the method of Nosaal (1952),
succinate by the method of Krebs (1937). Anaerobic manometrie experiments were carried
out under nitrogen, with yellow P in the centre well. CO, was measured by the "direct
method," bound CO, being released by 0-1 nil. of 5 N 11,80, (Umbreit et al, 1949).
AU concentrations reported in this paper are final iront-entrations. Unless otherwise stated,
the volume of reactants waa 3 ml. and the reactions were carried out for 1 hour at 38° C.
Austral. J. exp. Bid. (19S4), 32, pp. 8&~94.
86
PHILIP A. TRUDINGER
RESULTS.
Effect of dipicrylamine.
The effect of dipicrylamine on aspartic acid deamination is shown in Table 1.
Complete inhibition of the reaction was produced by 10"* M inhibitor. However, the action of dipicrylamine may not in fact be an immobilization of potassium. It can be seen from Table 1 that dipicrylamine was just as effective in
inhibiting the reaction in toluene-treated cells, in wliich no requirement for K +
can be shown (Trudinger, 1953).
TABLE 1.
Effect of dipicrylamine on aspartic acid deamination.
0-0025 M L-aspartate; 0-06li M K : Na phosphate, pH 7-2; 0-3-0-(i mg. dry wt. cells
(1-4 untreated, 5 toluene-treated).
Concentration of
dipicrylamine
0
10-* M
10-»M
10-'M
1
2
103
256
5
197
5
35
QNH,
3
266
8
210
4
5
225
8
94
217
123
8
112
1. Anaerobic, no additions. 2. Anaerobic + lO-'M glucose. 3. Anaerobic -j- 10"* M
adenodine. 4. Aerobic, no additions. 5. Aerobic, 2 x 1^"^ M M l
Effect of potassium.
The deamination of aspartie acid in sodium phosphate buffer was strongly
.stimulated by the addition of KCl (Table 2). The reaction rate at pH 7-0 was
almost optimal with 10"- M KCl, but small additional increases in activity were
observed as the KCl concentration was raised to 10"^ M (Fig. 4). Potassium
sulphate was slightly less effective than KCl, due to slight inhibition by the
sulphate ion. Rubidium salts were as effective as potassium; NaCI and LiaSO.)
were inert. A small stimulation by CsCl may have been due to contamination
by K+ or Ru + . K+ in the form of phosphate, borate and acetate was as effective
as KCl; potassium ions are therefore the active material.
TABLE 2.
Effect of KCl on deajnination in sodium phosphate.
0-0025 M L-aspartate; 0-066 M N a N a phosphEite, p H 7 - 0 ; co. 0-5 m g . d r y wt. cells.
E x p t . 1, A n a e r o b i c ; E x p t . 2, Aerobic.
^
QNH,
Expt.
1.
2.
Additiuna
None
Glucose 10-" M
Adenosine lO"'M
None
No KCl
+10-» M KCl
28
100
135 (107)*
282 (182)
143 (115)
300 (200)
126
249
* Figures in brackets are the increases in activity produced by glucose or adenosine.
DEAMINATION OF ASPARTIC ACID BY PROTEUS X-19
87
Stimulation by K+ was observed in the presenee or absence of glucose or
adenosine (Table 2). On a percentage basis, the effect of K+ on the "basic"
activity was greater, stimulation being 3- to 4-fold compared with ca. 2-fold in
the presence ol' glucose or adenosine. However, the activation of deamination
was further enhanced by K + .
Effect of sodiu7n.
In view of the marked effect of K+ on deamination rate, it was thought
that a pure potassium l)uffer would be preferable to the mixed sodium and
potassium usually employed. Under these conditions, however, low activities
were again observed which responded markedly to the addition of NaCl
(Table 3). Other members of the group I cationic series were ineffective. Na+
was active when added as chloride, sulphate, phosphate and acetate.
TABLE 3.
Effect of NaCl on deamination tn potassiwn pho3phate.
0-0025 M.L-nspartate; 0-060 M K: K phosphate, pH 7-0*; ca. 0-5 mg. dry wt. cells.
Expt. 1, Anaerobic; Expt. 2, Aerobic.
QNH,
Expt.
1.
2.
Additions
No NaCi
+ 1.0-»M NaCl
55
None
Glucose lO-'M
Adenofline 10-« M
None
181
264 (83)
272 (91)
143 (88)t
145 (90)
35
•MW
* Na* impurity in buffer was oa. 3 x 10"' Mt Figures in bracketa are the incTeasea in activity produced by glucose or adenosine.
In potassium buffer with added Na+, the basic activity was mueh higher
tlian that normally found in the K-Na buffers which have been generally used.
The concentration of Na+ required for optimal activity at pH 7-0 was ca.
lO'^M (Fig. 2), but, in contrast with K+, the activity decreased as the Na +
concentration was increased above 2 X 10"^ M. Variation in basic activity is
correlated with Na+ concentration rather than with, K: Na ratio (Table 4).
TABLE 4.
Effect of K:Na ratio on deamination activity.
0-0025 M L-aapartate; anaerobic.
Buffers (O-OiifiM, pll 7-0) made from mixtures of potassium and sodium phoBphatea to give
appropriate K:Na ratios.
QNH,
Additions
None
Glucose
None*
K:Na
-*
Molarity of Na —*
7:1
0-013
177
238
236
205
Aerobic,
3:1
0-023
177
235
266
205
1:1
0-053
122
221
221
198
1:3
0-085
81
223
231
204
1:7
0-094
85
210
220
202
88
PHILIP A. TRUDINGER
The actual increase in QNH^ produced by gflucose and adenosine was not
affeeted by the addition of Na+ (Table 3). Furthermore, the activity in their
presenee was not significantly altered by concentrations of Na+ sufficient to
decrease the basic activity hy up to 50 p.e. (Table 4).
Effect of pH,
It has been reported (Trudinger, 1951) that the pH for
optimal activity of whole cells
is between 6-8 and 7-2. As this
curve was obtained using the
usual Sorensen buffers made
from KH2PO4 and Na2HP04
(see Kolthoff and Rosenblum,
1937), the variation in K+ and
Na+ concentration at different
pH levels gives a distorted picture of the effect of hydrogen ion
concentration. The effect of pH
on deaminase activity was therefore re-investigated.
No potassium or sodium salts
were available which were entirely free from contamination,
and to offset any anomalies introduced by variation in this
contamination, NaCl or KCl respectively was added to the
appropriate buffer solutions so
that the contaminating metal
was present in equal concentration over the wliole pH range.
Fig. 1. Effeet of Na* and pH on deamination
in potaaaium buffer.
The level of impurity was
0.00:175 Rr L-aspartate; buffers 0-066 M (K» =
checked in each case on the flame 0-133
M); A and B, anaerobit^; C and D, anarrobic 4.10-* M glucose; E and P, aerobip,
spectrophotometer. No significNa* concentrations ( x 10' M)
ant concentration of metal was
A and C; 1, 10-7; 2, 5-7; .3, 2-7; 4, 0-7
introduced with the cells.
B and D: 1, 11-2; 2, 3-2; 3, 1-2
E: 1,25-7; 2,10-7; 3 , 5 - 7 ; 4 , 2 - 7 ;
The pH values of the various
5, 0-7
buffers were checked each day
F : 1, n - 2 ; 2, 6-3; 3, 3-2; 4, 1-2
and substrate was neutralized to
each respective pH. The change in pll during the reaction was never more
than 0-1 and usually less than 0- 05.
DEAMINATION OF AS1»ARTIC ACID BY PROTEUS X-lD
89
MVhen working at a pH above l-\i, small amounts of ammonia were expelled
from the reaction medium. At pll 8-5, this loss was often as high as 50 p.c. This
was overcome by using double side-armed tubes with substrate in one side-arm
and in the other 0-1 ml. 5 N H2SO4 to fix NH3. At the end of the reaction
I)eriod, the acid was tipped into the main compartment, bringing the pH down
to ca. 1-2 at which value aspartase is no longer active. The tubes were then
shaken for a further 10 minutes to ensure complete absorption of any free
ammonia in the tube. Under these conditions, 100 i).c. recovery of ammonia was
obtained at higii pH levels even when the reaction was run in vacuo.
The relation between activity and pH was found to depend on the ionic
composition of the medium.
Experiments in potassium buffer.
The pH range for optimal anaerobic activity in potassium borate containing
about O'Ol M Na+ was between pH 7*9 to 8-4, both in the presence and absence
of glucose (Fig. 1, B and D). In phosphate, high activity was obtained over a
mucli wider pH range, the rate of deamination being almost optimal from pH 7-5
ujtwurds (Fig. 1, A and C). Aerobically, the optimum was nearer pH 8-7 both
in jihosphate and borate (Fig. 1, E and F ) .
With lower Na+ concentrations,
tlie activity fell more rapidly as
the pH was decreased from the
optimum. This is due to a greater
Na+ requirement under the more
acid conditions (Fig. 2). Thus
anaerobically, optimal activity was
found with 10-- M Na+ at pli
7*95, while at pH 5-9 the rate was
still not maximal at 2*5 X
10"-' M Na+; aerobically, the sysrig. 2. Effect of Na* concentration ou
tem required for optimal activity,
deamiaatiou.
2-3 times as much Na+ at pH 5-9
0-00375 M L-aspartate; 0-06G M K: K phosphate.
as at pH 8-5. Concentrations of
A: Anaerobif, 1, pH 7-95; 2, pH 7-0; 3,
Na+ between 2-5 X 10""^ stimupH 5-9; 4, pH 5-35.
lated markedly at the alkaline pH
H: Aerobic, 1, pH 7-95; 2, pH 5-9; a, pH
u-35.
values, but not u nd er acidic
conditions.
About 5 X 10-2 JI j^a+ was required at pH 5-35. Tho inhibition of the
basic anaerobic deamination by higli concentrations of Na+ also became less as
the i)H was lowered; at pH 5-35, no inhibition was observed with 10"' M Na+.
A less pronounced increase in Na+ requirement was also found at pH values
above the optimal. Higher Na+ concentrations were required for optimal
activity, but deamination was increased significantly at Na+ levels whieh had
90
PHILIP A. TBUDINGER
no effect at acidic pH values. No work has been done at pH values above 9-5.
Results obtained anaerobically were essentially similar when glucose was replaced
by adenosine.
Experiments in sodium buffer.
In sodium buffers, two anaerobic pH optima were found in the absence of
glucose (Fig. 3, A and B); a relatively weak optimum appeared at pH 6-4-6-5
and a stronger one between pH 7*8 and 8-2. These peaks occurred regardless of
the K+ concentration of the buffer.
On the addition of glucose and 10"M K+, a broad optimui]i range, from
pH 7-0 to 8-0 in pliosphate and from
6-5 to 7*5 in borate, was observed.
At 10-^ M K + , however, two peaks
were observed in borate, the more
acidic of which disappeared with increasing K+ eoncentration. In phosphate, a flattening of the pH curve
was the only evidence of a second pH
optimum. In borate, increases in K+
concentration to 10"- M tended to
lower the pH optimum.
Two aerobic pH optima were also
evident in borate in the i)resenco of
low concentrations of K+ (Fig. .'J, F ) ;
as in the case of glueose-stimulated
anaerobic activity, increasing K""" to
10"- JI eliminated the more acidic
peak. In phosphate, there was no welldefined aerobic i)H optimum at the
lower pH values; the curves were
characterized by small but very sharj)
peaks between pH 7-35 and 7-95.
As in the case of sodium, low
concentrations of K+ had less pi-otiounced stimulatory effects with decreasing ])!:{. The results were less
striking than tiiose obtained with
Fig. 3. Effect of K* and pH on deamination
in sodium buffer.
sodium, and with the basic anaerobic
0-00375 M L-aapartate; buffers. 0-066 M system and aerobic system an increas(Na* = 0-13;i MJ. A and B, anaerobic, C and
D, anaerobic + 10"' M glucose; E and F, ed K+ requirement fur maximal
aerobic.
activity at acid pH values was not
K* concoutrations: Curves 1, lO"" M; 2, 10"* foimd (Fig. 4). The stimulated anM; 3, 10-» M.
DEAMINATION OF ASPARTIC ACID BY PROTEUS X-19
91
aerobic deamination showed greater K+ requirement for optimal activity at
acid pH values.
The basic anaerobic deamination in potassium buffer with 2 X 10'^ M Na+
i'ollowod a linear course to completion. In sodium buffer with added K + , tlio
jH'tlvity IVII witli time over an hourly period (Fig. 5).
30
rig. 4. Effect of K* concentration an deam in nti on.
AspiirtatP and buffers aa for Fig. 3.
A. Anaerobic, phosphate -j- 10-* M glucose; 1,
pH 7.n5; 2, p H 7 - 0 ; 3, pH 6-13.
B. Aerobic, phosphate; 1, pH 7-95; 2, pH
C. Aerobic, borate; 1, pH 8-05; 2, pH 6-51.
0-13.
^5
Minui»
«>
n g . 6. Course of deamiiintion in
low and high sodium ntediH.
250^1 aspartate; pH 7*2; Anaerobic.
Curve 1. K:K phosphate + 0-02
M NaCl.
Curve 2. Na:Na phosphate +
0-02 M KCl.
The effect of metal ions on the metabolism of fumarate.
Aspartase in toluene-treated cells or extracts was unaffected by the absence
of sodium or potasaium ions, suggesting that these ions act indirectly by affecting some metabolic process associated with the integrity of tlie cell wall. Therefore their effeet on fumarate metabolism was investigated (Table 5). Both
fermentation and oxidation of fumarate wei-e greatly reduced in the absence of
either K+ or Na+. However, in contrast to aspartate deamination, fumarate
metabolism was not inhibited by high coneentrations of sodium.
TABLE 5.
TJie effect of K* and Na* on fumarate metaholism.
(O'0G6 M phosphate, pH 7-2; 0-02 M fumarate; incubation, 60 mins.)
Medium
Na:Na phosphate
Na;Na phosphate f 0-02 M KCl
K:K phosphate
K:K phosphate + 0-02 M NaCl
Changes in fA.
Anaerobic
Pumarate
COj
Succinate
—260
—990
—200
—1030
+117
+510
+120
+530
+120
+510
+110
+560
Aerobic
O,
—95
—910
—117
—907
92
PHILIP A. TRUDIN'OER
DISCUSSION.
The effects on aspartie acid deamination of sodium, potassium and hydrogen
ions, reported in this paper, are related to the integrity of the cell wall. When
the membrane is partly disorganized by treatment with toluene, or I'reezinfj
and thawing (Trudinger, 1953) or when cell-extracts are used (unpublished
results), the requirement for K+ and Na+, and inhibition by high coneentrations of Na+, are no longer found. Further, the effect of pH becomes independent of the eationic constitution of the medium. A somewhat similar phenomenon has been described by Fleischmann and Schwarz (1037), who found that,
while Li+ and Na+ accelerate, and K+ arrests, methylene blue reduction by
yeast cells, these ions have no effect on the reduction of the dyestuff by maceration juicra.
Tn view of the well-known activation of fermentation by K+ (Farmer and
Jones, 1942; Muntz, 1947), it may well be that this ion exerts its effect indirectly
by affecting fermentative processes related to deamination. Since fumarate
metabolism is dependent on K+ it suggests that the rate of basic anaerobic
deamination may be governed by the rate of fumarate fermentation. The action
of Na+ is probably similar, since fumarate metabolism requires this ion also.
It lias been suggested already (Trudinger, 1951) that stimulation by glucose,
etc. ia related to its fermentation. The metabolism of glucose and adenosine by
Proteus is dependent on K+ but not on Na+ (unpublished results), which may
be the reason why stiimilation of deamination is increased by K+ in sodium
buffer but not by Na+ in potassium buffer.
One effect of glueose and other stimulators is to offset the inhibitory effect
of high concentrations of sodium ions. This may be related to the intraeellular
concentration of K+ by fermentation with a concurrent exclusion of Na+. Sueh
a phenomenon has been observed with red cells (Danowski, 1941; Pulver and
Verzar, 1941), yeast (Pulver and Verzar, 1940; Conway and O'Malley, 1946;
Rothstein and Enns, 194G: Scott et nl, 19r)l), brain cells (Dixon, 1949; Davies
and Krebs, 1952), and bacteria (Lieberwitz and Kupermintz, 1942; Oowie et al.,
1949; Roberts ei al, 1949).
The mechanism of sodium inliibition of the basic deamination is unknown.
Competitive antagonism of K+ by sodium has been observed in the growth of
laetie bacteria (MacLeod and Snell, 1948), but in tho case of aspartate deamination, inhibition is independent of the K+ concentration. Alternatively, it is
unlikely that sodium inhibits by preventing K+ accumulation by mass action
ainee (a) high concentrations of K+ do not antagonize Na+ although the latter
is equally essential for the deamination, and (b) above a certain level, increases
in Na+ concentration ])roducc no further inhibition of the deamination.
Although K+ and Na^ may stimulate deamination through their action on
fumarate metabolism, inhibition by high concentrations of Na+ cannot be thus
explained, since fumarate metabolism is not affected.
DEAMINATION OF ASPARTIO ACID BY PROTEUS X-19
93
The increased metal requirement with decreasing pH is probably due to
cationic exchange between K+ or Na+ and hydrogen ions {cf. Conway and
O'Malley, 1946; Rothstein and Enns, 1946; Conway and Brady, 1950; Robertson, 1950; Maeson and Lako, 1952). The phenomenon may be viewed in two
ways. Increases in metal ions may exclude hydrogen ions from tlie internal
environment, thus buffering the internal medium at a more favourable pH for
deamination; or an increase in H+ concentration may expel intracellular K"*"
and Na+ (observed by Eddy and Hinshelwood, 1950), necessitating higher
external concentrations of metallic ions to produce the internal levels required
for optimal metabolic activity. The absenee of inhibition by sodium at low pH
values may mean exclusion of this ion from the internal medium by hydrogen
ions.
Since either sodium or potassium is already present in the external medium
in high concentration as a constituent of the buffer, the latter postulate is considered most likely. Nevertheless, the tendency for the pH curves to "level out"
as the metal concentration is increased indicates that they may eontrol to some
extent the internal pH. Such a suggestion has been made by Eddy and Hinshelwood (1951), who demonstrated that, at adverse pH values, high concentration
of K+ stimulated glucose oxidation in E. coli, but not at physiological pH values.
However, deamination of asparagine and alanine did not respond to K+ at pH
values lower than optimal.
SUMMARY.
Intact Proteus X-19 requires sodium and potassium ions for maximal
deamination of aspartie acid. At pH 7, the optimal concentration for both ions
is ca. 0-01 M.
K+ coneentrations above 0*01 M increase slightly the rate of deamination,
but Na+ inhibits the basic anaerobic activity above 0-05 M.
Aerobie and stimulated anaerobic deamination are unaffected by high Na"^
concentrations.
The relation between pH and deamination is markedly affected hy the ionic
constitution of the medium.
Higher concentrations of metallic ions are required for optimal activation
at acid pH values than at more alkaline reactions. Small concentrations of K +
and Na+ which activate strongly at pll 7 and over may have no effect at pH 6.
Aeknowledgmcntn. The author is indebted to Professor M. L. Mitchell atid Dr. P. M.
Nossiil for help with tlip maiuiacript, and to the latter fur diacusalon and criticism of the
work preHtmted. Thanks are duo alao to the Physics Department, University of Adelaide, for
the use of the flame Bpeftrophotometer.
94
PHILIP A. TRUDINGER
REFERENCES.
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Conway, E. J. and Brady, T. G. (1950): Ibid., 47, p. 360.
Cowie, D. B., Roberts, R. B. and Roberts, E. Z. (1949): J. cell comp. Physiul.. 34, p. 243.
Danow.'ski, T. S. (1941); J. biol. Chem., i:i9, p. ()93.
Davies, R. E. and Krebs, H. A. (1952): Bioehem. J., 50, p. 25.
Dixon, K. C. (1949): Ibid., 44, p. 187.
Eddy, A. A. and Hinshehvood, C. (1950): Proc. Roy. Soc. I.oniJ., Scr. B., 130, p. 544.
Eddy, A. A. and Hlnahelwood, 0. (1951): Ibid., 138, p. 228.
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Maeaan, J. M. and Lako, E. (1952): Biorhim. Biophys. Acta, 9, p. 106.
Nossal, P. M. (1952): Biochem. J., 50, p. 349.
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Pulver, R. and VorzAr, F. (1941): Ihid., 24. p. 272.
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Robertson, R. N. (1950): Proc. Linn. Soc. N.S.W., 75, p. IV.
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